Formalin-fixed, paraffin-embedded (FFPE) tissue offers a vast source of biopsy specimens for which the clinical outcome is well documented and thus an optimal resource for retrospective studies (Lewis, F. et al. 2001; Yang et al. 2006). Access to and use of human specimens is an essential part of the cancer research and drug discovery infrastructure, enabling researchers to identify drug targets, develop lead compounds and understand drug metabolism. Research using human specimens can help predict drug response and toxicity, as well as short and long term clinical outcome. New technologies and information gained from mapping the human genome continue to fuel a growing need for researchers in academia and industry, for-profit and not-for-profit, to have access to good quality human specimens to expedite cancer drug discovery. Many different types of human specimens are required to support these studies: normal and malignant tissues, blood, other fluids, and the proteins, DNA and RNA that can be extracted from them.
Because all surgical procedures performed in the U.S. must obtain tissue samples for pathology diagnosis, primary sources for human specimens are hospital operating rooms and pathology laboratories. More than 160 million pathology specimens (most of them fixed tissue in wax blocks) are currently stored in the United States (Eiseman and Haga, 1999).
Tissue blocks are routinely fixed and embedded in paraffin, then sectioned with a microtome, and the sections affixed to microscope slides. The paraffin-embedded tissue sections have required dewaxing prior to analysis of nucleic acids to allow penetration by aqueous solutions.
For example, using a clean razor blade, FFPE sections have been scraped off slides and transferred into microfuge tubes for processing. The traditional method of paraffin removal involves organic extractions using xylene and graded alcohols. This procedure is time-consuming, cumbersome, and requires special handling, as xylene is a highly toxic chemical that emits noxious fumes. After 10 sections (60-100 microns/25-250 mm2) of FFPE specimens from the same tissue block are scraped off from glass slides into the tubes using a scalpel, one milliliter of hydrophobic solvent is added, e.g., xylene-containing EZDeWax™ (BioGenex, San Ramon, Calif., USA); see FIG. 1. After vortex mixing and incubating at room temperature for 5 min, the tissue samples are centrifuged in a microcentrifuge at 16,000×g for 2 min, and the supernatants removed. One milliliter 70% ethanol can be added to the samples, and the samples vortex mixed and centrifuged in a microcentrifuge at 16,000×g for 2 min. The sample wax is then extracted repeatedly into the xylene phase and the residue washed with 70% ethanol for two-five more times before continuing to the next step of tissue homogenate preparation or total RNA isolation (Yang et al., 2006).
The phase extraction dewaxing protocols are time consuming and laborious. The repeated handling, aspirations and tube transfers can result in non-quantitative harvests of the nucleic acids. The repeated vortexing and exposure to harsh solvents can cause sample degradation.
Additional problems exist in the quantitation of nucleic acids from preserved clinical specimens. For example, RNA quality can be affected by sample collection, formalin fixation and tissue processing. This can compromise, e.g., the ability to measure RNA in FFPE tissue blocks. The nucleic acids ultimately extracted from embedded clinical samples are often highly degraded and fragmented. Qualitative and quantitative assay errors often result when these extracts are evaluated by standard analytical techniques. What's more, incomplete extractions can introduce error into calculations, such as mRNA copy number determinations. One problem in measuring RNA from FFPE tissue blocks can be fragmentation of the RNA fragments, cross-linking, and base modifications induced by formalin-fixation procedures. Two processes that reduce the length of RNA molecules in formalin-fixed tissues are degradation and fragmentation (hydrolysis). RNA degradation can occur through enzymatic cleavage before the tissue encounters a fixative and is thus subject to the collection procedure of the samples. Fragmentation of RNA molecules can be caused by the formalin fixative and therefore varies substantially depending on formalin conditions employed (Lehmann U, Kreipe H: Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies, Methods 2001, 25:409-418). The exact causes for the fragmentation are not known, and thus it has been unclear how to solve this problem.
The current state-of-the-art technology for measuring RNA is quantitative PCR (QPCR). However, several recent reports comparing RNA quantification in frozen and FFPE tissues demonstrate that only 3-5% of RNA transcripts are available for detection by QPCR after formalin fixation (Bibikova M, Talantov D, Chudin E, Yeakley J M, Chen J, Doucet D, Wickham E, Atkins D, Barker D, Chee M, Wang Y, Fan J B: Quantitative gene expression profiling in formalin-fixed, paraffin-embedded tissues using universal bead arrays, Am J Pathol 2004, 165:1799-1807). This problem is independent of whether the reverse transcription step uses oligo-dT or random priming. A viable explanation for this problem is that reverse transcription and/or QPCR are severely affected by formalin mediated mono-methylolation of bases in RNA. Attempting to compensate for this problem, the expression of genes of interest has been normalized to internal housekeeping genes. However, this is often inadequate because adenines are more susceptible to alteration by formalin fixation and thus A/U rich sequences will be less accurately measured than G/C rich sequences (Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K: Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples, Nucleic Acids Res 1999, 27:4436-4443). Consequently, there will be gene specific differences in the efficiency and reproducibility of measuring mRNA in formalin-fixed tissues. In addition to its modification by formalin, the heavy fragmentation during the fixation and/or subsequent isolation process requires specialized primer design. Thus, there are severe limitations for PCR-based RNA measurements in formalin-fixed tissues. Alternative methods that are less sensitive to formalin-induced alterations are needed to improve the accuracy of RNA quantification (Bustin S A: Quantification of mRNA using real-time reverse transcription PCR(RT-PCR): trends and problems, J Mol Endocrinol 2002, 29:23-39; Bustin S A, Nolan T: Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction, J Biomol Tech 2004, 15:155-166 Gunther E C, Stone D J, Gerwien R W, Bento P, Heyes M P: Prediction of clinical drug efficacy by classification of drug-induced genomic expression profiles in vitro, Proc Natl Acad Sci USA 2003, 100:9608-9613).
Performance of quantitative PCR (QPCR) has faired poorly in quantitation of FFPE RNAs because it is generally limited to 75-85 bp amplicon size, and multiple pooled gene-specific primers are required (Cronin M, Pho M, Dutta D, Stephans J C, Shak S, Kiefer M C, Esteban J M, Baker J B: Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay, Am J Pathol 2004, 164:35-42). QPCR requires a much greater purity of RNA than the bDNA assay and thus more steps to process the samples prior to analysis compared to the bDNA technology. After dewaxing, the RNA needs to be digested with Proteinase K, isolated and submitted to 1-2 times of DNAase I treatment to remove DNA contamination. A second problem that affects RNA quantification by QPCR is the required reverse transcription step to convert mRNA sequences of interest to cDNA. This enzymatic reaction is impeded by formalin-induced base modifications, by secondary mRNA structure and by impurities in the RNA preparation. Factors inhibiting reverse transcription will vary amongst FFPE tissue blocks. Although, introduction of a high temperature heating step during PCR amplification steps may partially reverse some of the RNA base modifications, for many samples these modifications are irreversible. Older samples are often so impaired that a decrease in average QPCR signal is >90%, requiring more input RNA and increasing Ct values to 35-40 (Masuda N, Ohnishi T, Kawamoto S, Monden M, Okubo K: Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples, Nucleic Acids Res 1999, 27:4436-4443). With all these problems, QPCR has not been a satisfactory method of quantitating RNAs from FFPE samples.
In view of the above, a need exists for a faster and simpler way to harvest nucleic acids from embedded clinical tissue samples. It would be desirable to have a way to obtain nucleic acids from formalin fixed paraffin embedded samples without the use of hazardous solvents. The accuracy of nucleic acid analyses would benefit from a more quantitative and less damaging methods of nucleic acid extraction. Benefits can be obtained from methods to adjust analyses to take target degradation into consideration. The present invention provides these and other features that will be apparent upon review of the following.